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Amyloid plaques and neurofibrillary tangles of tau proteins are the two classic hallmarks of Alzheimer disease, but the connection between their two respective proteins—Aβ and tau—has remained mysterious. Now for the first time, a paper appearing July 22 in Cell details a molecular mechanism that links tau to Aβ toxicity at the synapse. Researchers led by Jürgen Götz and Lars Ittner at the University of Sydney, Australia, show that tau has a previously unknown role in the dendrite. Tau targets the Src kinase Fyn to the N-methyl-D-aspartic acid (NMDA) receptor, these authors report. This allows tau to mediate Aβ-induced excitotoxicity at the synapse. When tau is deleted or mistargeted in an AD model mouse, survival and memory improve to those of wild-type levels, although plaque burden and Aβ levels do not change. The same group shows, in a July 19 PNAS paper, that hyperphosphorylation of tau in a tau mouse model can be successfully treated with sodium selenate, leading to rescue of memory, motor performance, and neurogeneration. Both findings suggest promising new tau-based strategies for the treatment of dementias. Lars Ittner presented these data on July 15 in the very last session of the International Conference on Alzheimer’s Disease in Honolulu, Hawaii, where a diminished crowd of diehards gave it a favorable reception.

“I am very enthusiastic about th[is] paper for several reasons,” Lennart Mucke of the University of California San Francisco, wrote to ARF (see full comment below).

One clue came from the fact that tau protein contains a binding site for Fyn kinase. Fyn mills around at the post-synaptic density in wild-type mice, where it phosphorylates the 2b subunit of the NMDA receptor (NR2b). This strengthens the interaction of the NMDA receptor with the post-synaptic density protein 95 (PSD-95), and leads to excitotoxic downstream signaling. Overexpression of Fyn increases Aβ toxicity (see Chin et al., 2004 and Chin et al., 2005).

First authors Ittner and Yazi Ke looked for Fyn in a tau knockout (KO) mouse, and found it to be reduced by two-thirds at the synapse. This indicates that tau plays an important role in targeting Fyn to the synapse, although some Fyn arrives at the synapse independently of tau. Ittner and colleagues then generated a transgenic mouse that expresses a truncated version of the tau protein (Δtau74) under a neuronal promoter. The truncated version lacks microtubule-binding domains and cannot form aggregates, but includes the amino-terminal projection domain with its binding site for Fyn kinase. Truncated tau localizes to the membrane of the cell body, but is not present in dendrites, and so is incapable of targeting Fyn to the synapses themselves.

Ittner and colleagues found that in the Δtau74 transgenic mouse, Fyn was down by three-quarters at the synapse, despite the presence of endogenous tau. It turns out that truncated tau acts as a dominant-negative mutation by competing with endogenous tau to bind Fyn and mistarget it. As evidence of this, in the Δtau74 mouse, co-immunoprecipitation with Fyn mostly pulls down truncated tau, not endogenous tau. As might be expected with less Fyn at the synapse, in both Δtau74 and tau-null mice there was less phosphorylation of NR2b, and fewer NR subunits co-immunoprecipitated with PSD-95, indicating a weaker interaction. Importantly, Δtau74 and tau-null mice were less susceptible to seizures, which result from overstimulation. Despite these changes, synaptic currents were normal in both tau mutant strains.

The authors then looked at what effect these changes in tau might have on AD by crossing the two tau strains with an AD mouse model (APP23), both independently and in combination. Both tau deletion and transgenic tau independently improved the memory of APP23 mice to wild-type levels. Both tau double-crosses also survived longer than the APP23 mice, which have a premature mortality phenotype, and in fact, the combination of transgenic tau with endogenous tau deletion fully rescued survival. The tau crosses also reduced excitotoxicity in the APP23 mice, decreasing the severity of seizures. Significantly, Aβ levels and plaque load were unchanged in these double-crosses, indicating that tau acts downstream of Aβ. Importantly, Ittner and colleagues used a different tau KO strain and different AD mouse strain than the 2007 study by Roberson et al., and yet they saw the exact same effect, demonstrating that this finding is robust and not dependent on a particular mouse strain.

These results implied that the NMDA receptor-PSD-95 interaction is a crucial feature of Aβ-induced excitotoxicity. To test this, the authors made use of a small peptide, Tat-NR2B9c, which has been shown to interfere with the NMDA receptor PSD-95 interaction and is already known to reduce excitotoxicity in a mouse model of ischemia (see Aarts et al., 2002). When primary neuronal cultures from wild-type mice were treated with this peptide, the neurons became more resistant to cell death induced by Aβ treatment. The authors then used osmotic mini-pumps to infuse the peptide into APP23 mice for eight weeks. Treated mice had fewer seizures, their memory improved, and their survival returned to near wild-type levels, even several months after treatment.

The results suggest several therapeutic possibilities, Götz said. Reducing tau levels can improve symptoms in AD mouse models, and therefore might be beneficial in people. Another exciting avenue might be to treat with a peptide or, better yet, small molecule that disrupts the NR2b-PSD-95 interaction, or with the tau projection domain, since these interventions weaken excitotoxicity without interfering with normal synaptic transmission. It is especially intriguing that a narrow therapeutic window of peptide treatment led to long-term protection, Götz said, and one of the more fascinating questions he intends to pursue is what might be the biological basis of that window. Other questions include discovering how Aβ acts to exert toxicity. Does it act extracellularly or intracellularly? The authors would also like to investigate whether the interaction between tau and PSD-95 is direct or indirect, Götz said.

Previous research had shown that Aβ can accelerate an existing tau pathology, but “these new findings show that Aβ toxicity is dependent on the presence of tau, and provide a molecular mechanism for that,” Götz said. The authors have also demonstrated a critical role for tau in dendrites, in contrast to the traditional conception of tau as an axonal protein. The findings are not in disagreement with previous work on tau, Götz said. “I believe there are different cellular compartments of tau, because tau most likely is not free; it exists always bound to something.” The majority of tau is bound to microtubules, and when it becomes hyperphosphorylated, it detaches from microtubules and forms tangles in cell cytoplasm (see Geschwind, 2003). By contrast, the dendritic pool of tau is small, Götz said.

In the second paper, the authors focused on hyperphosphorylated tau, rather than dendritic tau. First author Janet van Eersel used two tau mutant mouse models: pR5 mice that develop neurofibrillary tangles (NFTs) at six months, and K3 mice that develop parkinsonism and memory impairment. The authors showed that treatment with the small compound sodium selenate reduced tau phosphorylation and eliminated NFTs in both tau mouse models, in vitro and in vivo. Selenium is a crucial trace element in brain. Some forms, such as sodium selenite, are associated with toxicity; however, the authors found no toxic effects from sodium selenate, a more oxidized form of selenium, after four months of treatment.

Protein phosphatase 2A (PP2A) is a major phosphatase responsible for tau dephosphorylation, and both the level and activity of PP2A are down in the AD brain. The authors found that selenate treatment greatly increased the amount of PP2A that co-immunoprecipitated with tau, implying that selenate stabilizes the tau-PP2A complexes, allowing the phosphatase to more readily dephosphorylate tau. To test this idea, van Eersel and colleagues crossed the pR5 mouse with the Dom5 transgenic mouse, which expresses a dominant-negative form of PP2A, and demonstrated that selenate treatment was no longer able to reduce tau phosphorylation and NFTs.

Since sodium selenate mitigates tau pathologies in several tau model mice strains, it is a promising compound for drug development, Götz said. “It amazes me how this compound works, and you see it works on several levels,” Götz said, explaining that it not only reduces tau phosphorylation and tangle formation, but it also ameliorates motor deficits in the K3 mice and memory impairment in the pR5 mice, and prevents neurodegeneration of cerebellar basket cells.—Madolyn Bowman Rogers

Comments

I am very enthusiastic about the paper by Ittner et al. for several reasons. First, it confirms the highly protective effects of tau reduction we observed in hAPP-J20 mice (Roberson et al., 2007 and Palop et al., 2007) in another APP transgenic line with a solid AD-like phenotype and on an independent tau knockout strain. As in our lines, tau reduction rescued memory and longevity in APP23 mice without changing Aβ levels or plaque loads. This kind of reproducibility underlines the robustness of the tau reduction effects and is reassuring to me, especially in light of a recent report suggesting that tau ablation changes Aβ levels and plaque loads in opposite directions and has adverse effects in the Tg2576 model (Dawson et al., 2010).

Taken together, these findings strongly suggest that modulating tau, its interaction with Fyn, or key proteins involved in or affected by this interaction may be of therapeutic benefit in AD. It remains possible, though, that other mechanisms also contribute to the excito-protective effects of tau reduction, including changes in presynaptic terminals or in the axonal transport of cargoes supporting synaptic functions.

In this manuscript, Ittner and colleagues showed that tau has a role in Aβ toxicity, which may be different from the role of tau on microtubules. Interaction of tau and Fyn is required for stabilizing the NR2/PSD95 complex. Reduction of tau, or interfering with the interaction of tau and Fyn, rescued the premature death and memory deficit in the APP Tg mouse. The results are very interesting, and suggest tau as an attractive drug target for AD therapy.

The physiological role of tau has been thought of as microtubule stabilization. However, the tau gene-deficient mouse did not show much evidence of brain dysfunction. Recently, the results of crossbreeding tau-deficient mice with the GSK3β overexpression or the APP overexpression mouse were reported. Reduction of tau level rescued both the impairment of LTP caused by GSK3β overexpression, and the memory deficits caused by APP overexpression (Gomez de Barreda et al., 2010; Roberson et al., 2007). These reports and the paper by Ittner et al. suggest that tau may have some roles in the synapse in addition to stabilizing microtubules. The extent of memory impairment in APP Tg mice depends on hippocampal LTP level. Tau is involved in synaptic plasticity, and deficiency of tau rescues LTP in the APP Tg mouse. I am not sure whether the destabilization of NR2/PSD95 in the tau knockout mouse can explain the attenuation of LTP level in the APP Tg mouse.

There are two different tau-deficient mouse lines. One is a simple tau gene knockout, and the other involves the replacement of exon 1 of the tau gene by EGFP cDNA. The former two reports used the tau gene knockout mouse, and Ittner used the EGFP mouse as a tau-deficient mouse. We may need to give careful consideration to this difference.

In this interesting paper, Ittner et al. go a long way towards explaining why tau protein is required for the synaptotoxic effects of β amyloid in transgenic mouse models of β amyloidosis. Previous work, mostly by Lennart Mucke and colleagues (Roberson et al., 2007; Chin et al., 2004, 2005), described the relevance of tau and the tyrosine kinase Fyn for mediating the toxicity of β amyloid. Earlier work by Gloria Lee and colleagues had shown that a PXXP motif (residues 175-178) in the amino-terminal half of tau binds to the SH3 domain of Fyn (Lee et al., 1998). Fyn phosphorylates tau at tyrosine residue 18 (Lee et al., 2004). The new work shows that tau is required for the correct post-synaptic localization of Fyn. Through phosphorylation of the NR2 subunit of the NMDA receptor, Fyn strengthens the interaction between NMDA receptors and the post-synaptic density protein PSD-95, which is required for nitric oxide production and downstream neurotoxicity. In the absence of tau and/or following overexpression of a dominant-negative tau fragment encompassing amino acids 1-255, Fyn was mislocalized and toxicity reduced, resulting in the prevention of the memory deficits and the premature death that are characteristic of transgenic mice from line APP23. These experiments reveal a novel function for wild-type tau, which is independent of its ability to interact with microtubules. A role in dendrites was unexpected, because tau is largely an axonal protein. From the above, it follows that mice doubly transgenic for mutant APP and wild-type tau will exhibit increased synaptotoxicity, whereas mice expressing mutant APP, in conjunction with wild-type tau lacking residues 175-178, will show reduced toxicity.

It is well established that β amyloid promotes the phosphorylation and aggregation of tau in the brain of transgenic mice expressing mutant, but not wild-type, human tau (Lewis et al., 2001; Boutajangout et al., 2004; Bolmont et al., 2007). This effect may be mediated by the serine/threonine protein kinase glycogen synthase kinase-3β (GSK3β) (Terwel et al., 2008; Hernández et al., 2010) or the C-terminus of heat-shock protein 70-interacting protein (CHIP) (Oddo et al., 2008). A similar phenomenon has been described in mice doubly transgenic for the Danish mutant form of human BRI2 and human mutant P301S tau, where the presence of abundant extracellular deposits of Dan-amyloid in cerebral cortex promoted tau phosphorylation and aggregation (Coomaraswamy et al., 2010). It will be interesting to see how these findings tie in with the work of Ittner et al., which appears not to involve phosphorylation or aggregation of tau. It is looking increasingly likely that reducing tau levels in brain will be therapeutically beneficial.

The important result by Ittner et al. that post-synaptic targeting of the Src kinase Fyn depends on tau should also be relevant to p75-mediated Aβ toxicity. The observed prevention of Aβ toxicity in APP23 mice with absent or truncated tau could, in part, be due to diminished p75 activity since Src kinases are required for p75 activation by Aβ aggregates (Egert et al., 2007).

I agree with Lennart Mucke, Akihiko Takashima, and Michel Goedert that this is a major opus by Ittner and Goetz and coworkers, and will become seminal in the long-standing question of how amyloid and tau are related to each other in the pathogenic processes in AD. The amyloid-tau relation is central by definition, as well as pathologically diagnostic for AD. Moreover, I approach the age where the matter becomes personally more and more important to be solved sooner rather than later. The issues at hand have separated "baptists" and "tauists" for too long, and for no apparent reason. I, at least, have adhered to both convictions over the last 20 years without too much negative consequences. I therefore welcome the Ittner study also in this respect.

Whether Fyn is "the" missing link in AD needs, and deserves, careful consideration, but this study will undoubtedly impact the field for some time to come. The data presented were dug out of an impressive number of cellular and mouse models by a wide range of technologies. Typical for the better studies is that they stir up more questions and have many implications that I have yet to come to full terms with, given the mass of finer details uncovered by these findings. Here, I planned to restrict myself to aspects and questions closest to our own scientific interests, i.e., the synaptic effects of tau and its phosphorylation in vivo (by GSK3 mainly), and the relation to amyloid in various mouse models, as we recently reviewed (Jaworski et al., 2010).

First, the evidence for NR2b phosphorylation by Fyn is convincing, but I wondered about the distinction between "electric" signals at the synapse (graphically not too well depicted in Fig. 7), namely, the classic ESPCs that are normal, as opposed to the "excitotoxic" signals that are strongly affected. What are these latter signals, and what is their function in wild-type mice under normal physiological conditions? These are described for wild-type mice in Figure 7, panel A—but perhaps the caption for that panel should be "APP23 mice." The data imply at least two pools of NR2bs in the same synapse, containing sub-subtypes, truncated, or otherwise modified or adapted subunits, extra-synaptic or tethered.

Teleologically, the most active synapses that produce the most Aβ peptides (for the sake of simplicity, I used the term "Aβ" throughout to indicate all molecular forms and complexity of all the amyloid peptides) must then also be the most vulnerable to this novel mechanism. How do active synapses counteract the inherent Aβ-mediated excitotoxicity? Is this the price to pay for an "LTP-ed" synapse?

A most mind-troubling issue, even an enigma, for me, is why the protein tau remains labeled by some of the most ardent tauists as "an axonal protein," c.q. specific axonal marker? Because we are interested in the role of tau in those brain regions that are struck in AD, we deal with glutamatergic synapses that are located on dendritic spines. I permanently force my Ph.D. students and coworkers to search the literature for evidence that tau is present not only pre-synaptically in axons, but also post-synaptically in dendritic spines—so far unequivocal proof is lacking. We do see, without many problems, mouse protein tau in dendritic spines in primary neuron cultures, and definitely in transfected neurons expressing human tau. Apparently, extending this to mouse brain in vivo, i.e., demonstrating that tau in spines in wild-type mouse brain sections, is technically (too) demanding, but must be done sooner than later….

In another vein, and raising further questions, is the notion that microtubules are not permanently but dynamically based in dendritic spines (Jaworski et al., 2009). This leads us to another dissociation: If protein tau is present in dendritic spines, which we never doubted, it cannot be bound to microtubules. That leaves it free to become bound to the PSD, proposed and implicated here by Ittner et al., or become sequestered to the actin network—that other important post-synaptic scaffold in spines.

A most intriguing question, raised but discussed somewhat "en passant" by Ittner et al., is, How does protein tau get into the dendrites and post-synaptic compartments, i.e., spines, in the first place? Besides the "piggy-back transport of Fyn," the findings highlight a major “normal” physiological function of protein tau in dendrites, which could potentially become more important for neuroscience than for the AD field (this thought illustrates nicely the narrow boundary between physiology and pathology, a concept instilled in my brain in my early Ph.D. student days!). There is, nevertheless, an important pathological flipside even to this dendritic tau: the dramatic neuropil tauopathy in AD, which according to experts is quantitatively an order of magnitude more important than neurofibrillary tangles in the soma (Mitchell et al., 2000). Is that exclusively axonal and pre-synaptic—or what is the contribution of dendritic post-synaptic compartments? In this respect, the statement by Ittner et al that "...levels of tau in the dendritic compartment are much lower than in axons,..." needs to be taken cautiously and verified by proper quantitative methods—if at all possible.

We used an AAV-tau.255 vector to express the same truncated version of tau, which, in contrast to full-length tau.4R and tau.P301L, was not neurotoxic. The lack of toxicity correlated with the fact that tau.255 was largely retained to the cell soma, as opposed to full-length tau that located to dendrites (see, e.g., Fig. S5D in Jaworski et al., 2009). We proposed that the missing microtubule binding domain (MTBD) and the inherent lack of transport over microtubules (MTs) prevented tau.255 from reaching the post-synaptic compartment in the dendritic spines. Interestingly, we observed increased phosphorylation of tau residues that constitute epitope AT180, i.e., S231/S235, which align closely with the 7th PXXP motif (P233-K-S-P235) that binds Fyn (Lee et al., 1998; confirmed here by Ittner et al.). Hardly accidental is then the fact that the AT180 epitope is typically generated by GSK3, which is the subject of our current efforts to understand the contribution of these kinases to the physiology and pathology of APP and protein tau (Terwel et al., 2008).

Importantly, in our AAV-based model, even wild-type tau was neurotoxic, causing rapid and extensive degeneration of CA1/2 pyramidal neurons in wild-type mice, i.e., in absence of human amyloid peptides, and without formation of large tau aggregates in soma or neuropil. Unfortunately, Ittner et al. did not analyze or discuss the eventual neurodegeneration they might have observed in their combined mouse models. Combining the novel data with previously published data leads me to conclude that Aβ causes excitotoxicity and provokes seizures, eventually causing premature death, but does not cause neuronal cell death—an observation made in many single transgenic APP mouse models.

The novel, documented NR2b-Fyn-mediated mechanism depends on transport of Fyn by endogenous murine protein tau into the post-synaptic compartment of dendritic spines. As stated above, the transport of Fyn appears to be "piggy-backed" on protein tau into dendritic processes, which inevitably could or even must result in phosphorylation of tau at Y18 (Lee et al., 2004). Does this phosphorylation come into play in the mechanism? Moreover, Fyn is transported by full-length tau over the dendritic MT system involving the tau-MTBD domain, which implicates this is not a novel function for tau, but "a novel route." This raises again other questions as the MT system in dendrites is not polarized like the axonal MT system. How tau manages to overcome the dendritic "traffic jam" is open for experimentation, which I am sure some of us are doing already.

I agree with Akihiko Takashima that GSK3 must be accounted for in the amyloid-tau equation. I beg to disagree with him regarding the statement that "Reduction of tau rescued…the impairment of LTP caused by GSK3β overexpression…." as LTP was not measured, but the spatial cognition task in the water maze was (Gomez de Barreda et al., 2010). Moreover, the data show that tau-/- did not rescue the GSK3-imposed phenotype, as the Tet/GSK3β mice did not differ significantly from Tet/GSK3β+tau-/- mice (Fig. 2 in Gomez de Barreda et al., 2010).

With regard to GSK3, I commented on this forum on a closely related study on amyloid-induced axonal transport problems involving NMDAR implicating GSK3 in the mechanism (Decker et al., 2010). Obviously, amyloid, tau, and GSK3 play on the same team more often than not, both at the physiological or pathological side of the brain.

Technically, I am not convinced that an issue remains in terms of tau-/- mice. I agree fully with Lennart Mucke that Ittner et al. reassures us that APP-induced deficits are mitigated in a tau-/- background, even in different parental strains (Roberson et al., 2007; Ittner et al., 2010). Science is more often than not reproducible and therefore pleasing and rewarding!

Allow me to conclude by stating that all commentators agree with Ittner and colleagues that protein tau is an attractive study target—and a promising drug target for primary and secondary tauopathies.